Tau-crystallin/alpha-enolase: one gene encodes both an enzyme and a lens structural protein.

tau-Crystallin has been a major component of the cellular lenses of species throughout vertebrate evolution, from lamprey to birds. Immunofluorescence analysis of the embryonic turtle lens, using antiserum to lamprey tau-crystallin showed that the protein is expressed throughout embryogenesis and is present at high concentrations in all parts of the lens. Partial peptide sequence for the isolated turtle protein and deduced sequences for several lamprey peptides all revealed a close similarity to the glycolytic enzyme enolase (E.C. 4.2.1.11). A full-sized cDNA for putative duck tau-crystallin was obtained and sequenced, confirming the close relationship with alpha-enolase. Southern blot analysis showed that the duck genome contains a single alpha-enolase gene, while Northern blot analysis showed that the message for tau-crystallin/alpha-enolase is present in embryonic duck lens at 25 times the abundance found in liver. tau-Crystallin possesses enolase activity, but the activity is greatly reduced, probably because of age-related posttranslational modification. It thus appears that a highly conserved, important glycolytic enzyme has been used as a structural component of lens since the start of vertebrate evolution. Apparently the enzyme has not been recruited for its catalytic activity but for some distinct structural property. tau-Crystallin/alpha-enolase is an example of a multifunctional protein playing two very different roles in evolution but encoded by a single gene

Stromal Edema in Klf4 Conditional Null Mouse Cornea Is Associated with Altered Collagen Fibril Organization and Reduced Proteoglycans

PURPOSE. Klf4, one of the highly expressed transcription factors in the mouse cornea, plays an important role in maturation and maintenance of the ocular surface. In this study, the structure and proteoglycan composition of the Klf4 conditional null (Klf4CN) corneal stroma was investigated, to further characterize the previously reported Klf4CN stromal edema. METHODS. Collagen fibril spacing and diameter were calculated from scattering intensity profiles from small angle synchrotron x-ray scattering patterns obtained across the cornea along a vertical meridian at 0.5-mm intervals. Collagen fibril organization and proteoglycans were visualized by electron microscopy (EM), with or without the cationic dye cuprolinic blue. Proteoglycans and glycosaminoglycans were further analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and immunoblot analysis. Q-RT-PCR was used to measure the transcript levels. RESULTS. In the central cornea, the average collagen interfibrillar Bragg spacing increased from 44.5 nm (SD ±1.8) in wild-type to 66.5 nm (SD ±2.3) in Klf4CN, as measured by x-ray scattering and confirmed by EM. Mean collagen fibril diameter increased from 32 nm (SD ±0.4) in wild-type to 42.3 nm (SD ±4.8) in Klf4CN corneal stroma. Downregulation of proteoglycans detected by EM in the Klf4CN stroma was confirmed by FACE and immunoblot analysis. Q-RT-PCR showed that, whereas the Klf4CN corneal proteoglycan transcript levels remained unchanged, matrix metalloproteinase (MMP) transcript levels were significantly upregulated. CONCLUSIONS. The Klf4CN corneal stromal edema is characterized by increased collagen interfibrillar spacing and increased diameter of individual fibrils. The stroma also exhibits reduced interfibrillar proteoglycans throughout, which is possibly caused by increased expression of MMPs

Regulation of Mouse Small Heat Shock Protein αb-Crystallin Gene by Aryl Hydrocarbon Receptor

The stress-inducible small heat shock protein (shsp)/αB-crystallin gene is expressed highly in the lens and moderately in other tissues. Here we provide evidence that it is a target gene of the aryl hydrocarbon receptor (AhR) transcription factor. A sequence (−329/−323, CATGCGA) similar to the consensus xenobiotic responsive element (XRE), called here XRE-like, is present in the αBE2 region of αB-crystallin enhancer and can bind AhR in vitro and in vivo. αB-crystallin protein levels were reduced in retina, lens, cornea, heart, skeletal muscle and cultured muscle fibroblasts of AhR−/− mice; αB-crystallin mRNA levels were reduced in the eye, heart and skeletal muscle of AhR−/− mice. Increased AhR stimulated αB-crystallin expression in transfection experiments conducted in conjunction with the aryl hydrocarbon receptor nuclear translocator (ARNT) and decreased AhR reduced αB-crystallin expression. AhR effect on aB-crystallin promoter activity was cell-dependent in transfection experiments. AhR up-regulated αB-crystallin promoter activity in transfected HeLa, NIH3T3 and COS-7 cells in the absence of exogenously added ligand (TCDD), but had no effect on the αB-crystallin promoter in C2C12, CV-1 or Hepa-1 cells with or without TCDD. TCDD enhanced AhR-stimulated αB-crystallin promoter activity in transfected αTN4 cells. AhR could bind to an XRE-like site in the αB-crystallin enhancer in vitro and in vivo. Finally, site-specific mutagenesis experiments showed that the XRE-like motif was necessary for both basal and maximal AhR-induction of αB-crystallin promoter activity. Our data strongly suggest that AhR is a regulator of αB-crystallin gene expression and provide new avenues of research for the mechanism of tissue-specific αB-crystallin gene regulation under normal and physiologically stressed conditions

Diverse Roles of Eph/ephrin Signaling in the Mouse Lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells

The Three-Dimensional Distribution of αA-Crystalline in Rat Lenses and Its Possible Relation to Transparency

Lens transparency depends on the accumulation of massive quantities (600–800 mg/ml) of twelve primary crystallines and two truncated crystallines in highly elongated “fiber” cells. Despite numerous studies, major unanswered questions are how this heterogeneous group of proteins becomes organized to bestow the lens with its unique optical properties and how it changes during cataract formation. Using novel methods based on conical tomography and labeling with antibody/gold conjugates, we have profiled the 3D-distribution of the αA-crystalline in rat lenses at ∼2 nm resolutions and three-dimensions. Analysis of tomograms calculated from lenses labeled with anti-αA-crystalline and gold particles (∼3 nm and ∼7 nm diameter) revealed geometric patterns shaped as lines, isosceles triangles and polyhedrons. A Gaussian distribution centered at ∼7.5 nm fitted the distances between the ∼3 nm diameter gold conjugates. A Gaussian distribution centered at ∼14 nm fitted the Euclidian distances between the smaller and the larger gold particles and another Gaussian at 21–24 nm the distances between the larger particles. Independent of their diameters, tethers of 14–17 nm in length connected files of gold particles to thin filaments or clusters to ∼15 nm diameter “beads.” We used the information gathered from tomograms of labeled lenses to determine the distribution of the αA-crystalline in unlabeled lenses. We found that αA-crystalline monomers spaced ∼7 nm or αA-crystalline dimers spaced ∼15 nm center-to-center apart decorated thin filaments of the lens cytoskeleton. It thus seems likely that lost or gain of long-range order determines the 3D-structure of the fiber cell and possible also cataract formation

Quantitative sequence-function relationships in proteins based on gene ontology

<p>Abstract</p> <p>Background</p> <p>The relationship between divergence of amino-acid sequence and divergence of function among homologous proteins is complex. The assumption that homologs share function – the basis of transfer of annotations in databases – must therefore be regarded with caution. Here, we present a quantitative study of sequence and function divergence, based on the Gene Ontology classification of function. We determined the relationship between sequence divergence and function divergence in 6828 protein families from the PFAM database. Within families there is a broad range of sequence similarity from very closely related proteins – for instance, orthologs in different mammals – to very distantly-related proteins at the limit of reliable recognition of homology.</p> <p>Results</p> <p>We correlated the divergence in sequences determined from pairwise alignments, and the divergence in function determined by path lengths in the Gene Ontology graph, taking into account the fact that many proteins have multiple functions. Our results show that, among homologous proteins, the proportion of divergent functions decreases dramatically above a threshold of sequence similarity at about 50% residue identity. For proteins with more than 50% residue identity, transfer of annotation between homologs will lead to an erroneous attribution with a totally dissimilar function in fewer than 6% of cases. This means that for very similar proteins (about 50 % identical residues) the chance of completely incorrect annotation is low; however, because of the phenomenon of recruitment, it is still non-zero.</p> <p>Conclusion</p> <p>Our results describe general features of the evolution of protein function, and serve as a guide to the reliability of annotation transfer, based on the closeness of the relationship between a new protein and its nearest annotated relative.</p

The Congenital Cataract-Linked G61C Mutation Destabilizes γD-Crystallin and Promotes Non-Native Aggregation

γD-crystallin is one of the major structural proteins in human eye lens. The solubility and stability of γD-crystallin play a crucial role in maintaining the optical properties of the lens during the life span of an individual. Previous study has shown that the inherited mutation G61C results in autosomal dominant congenital cataract. In this research, we studied the effects of the G61C mutation on γD-crystallin structure, stability and aggregation via biophysical methods. CD, intrinsic and extrinsic fluorescence spectroscopy indicated that the G61C mutation did not affect the native structure of γD-crystallin. The stability of γD-crystallin against heat- or GdnHCl-induced denaturation was significantly decreased by the mutation, while no influence was observed on the acid-induced unfolding. The mutation mainly affected the transition from the native state to the intermediate but not that from the intermediate to the unfolded or aggregated states. At high temperatures, both proteins were able to form aggregates, and the aggregation of the mutant was much more serious than the wild type protein at the same temperature. At body temperature and acidic conditions, the mutant was more prone to form amyloid-like fibrils. The aggregation-prone property of the mutant was not altered by the addition of reductive reagent. These results suggested that the decrease in protein stability followed by aggregation-prone property might be the major cause in the hereditary cataract induced by the G61C mutation